Rupture of the anterior cruciate ligament (ACL) occurs in over 100,000 patients each year. It cannot heal spontaneously. As neither surgical repair nor non-surgical treatments provide a reliably satisfactory clinical outcome, ACL ruptures remain a pressing orthopaedic problem. The research described in this proposal is directed towards improving the repair of the ACL through the application of novel biological approaches. These are based upon recent data suggesting that, contrary to the received view, the ruptured ACL mounts a healing response when a reinforced collagenous hydrogel is interposed between the severed ends of the ligament. Critical to this response is the migration of cells from the damaged ligament into the hydrogel. Preliminary data suggest that these are not representative of ACL cells as a whole, but constitute a distinct population which shares many of the properties of mesenchymal stem cells (MSCs). To achieve a clinically useful repair, these cells must divide, differentiate into ligament cells and lay down a matrix of sufficient mechanical strength. The experiments described in this proposal address these fundamental issues using in vitro and in vivo porcine models. In Specific Aim 1, the cells that migrate from the damaged ACL will be recovered from the hydrogel and investigated by biological and immunological criteria to determine to what degree they share the properties of MSCs. Their location within the ACL will be determined by immunohistochemistry. In Specific Aim 2, the responses of therse cells to a selected sub-set of growth factors will be determined. These factors are: FGF-2, BMP-12, BMP-13 and TGF-beta1. Both adenovirus- mediated gene transfer and, where possible, recombinant proteins will be evaluated in monolayer culture and in an in vitro "gap" model of ACL injury and repair. In Specific Aim 3, a full thickness, ACL rupture model in the pig will be used to determine the mechanical strength that is achievable when a gene-laden, reinforced hydrogel is used to repair a clinically-relevant defect. MRI, histology and immunohistochemistry will also be used to evaluate the healed ligament. These studies will advance our knowledge of the biology of the injured ACL and suggest novel, biologically-based approaches to healing.